The Relationship between Dendritic Branch Dynamics and CPEB-Labeled RNP Granules Captured in Vivo.

Bestman JE, Cline HT - Front Neural Circuits (2009)

Bottom Line:
In dendrites, the distributions of the active and inactive CPEB-containing RNP granules do not differ; the RNP granules are dense and their positions do not correlate with sites of rapid dendritic branch dynamics or the eventual fate of the dendritic branches.Because CPEB's sensitivity to activity-dependent signaling does not alter its dendritic distribution, it indicates that active sites in the dendritic arbor are not targeted for RNP granule localization.Nevertheless, inactive CPEB accumulates in granules in terminal dendritic branches, supporting the hypothesis that upon activation CPEB and its mRNA cargo are released from granules and are then available for dendritic translation.

ABSTRACTCytoplasmic Polyadenylation Element Binding protein (CPEB) is an RNA binding protein involved in dendritic delivery of mRNA and activity-dependent, polyadenylation-induced translation of mRNAs in the dendritic arbor. CPEB affects learning and memory and impacts neuronal morphological and synaptic plasticity. In neurons, CPEB is concentrated in ribonucleoprotein (RNP) granules that distribute throughout the dendritic arbor and localize near synapses, suggesting that the trafficking of RNP granules is important for CPEB function. We tagged full-length CPEB and an inactive mutant CPEB with fluorescent proteins, then imaged rapid dendritic branch dynamics and RNP distribution using two-photon time-lapse microscopy of neurons in the optic tectum of living Xenopus laevis tadpoles. Though the inactive CPEB mutant transports mRNA in the dendritic arbor, its expression interferes with CPEB-dependent translation because it is incapable of activity-triggered mRNA polyadenylation. In dendrites, the distributions of the active and inactive CPEB-containing RNP granules do not differ; the RNP granules are dense and their positions do not correlate with sites of rapid dendritic branch dynamics or the eventual fate of the dendritic branches. Because CPEB's sensitivity to activity-dependent signaling does not alter its dendritic distribution, it indicates that active sites in the dendritic arbor are not targeted for RNP granule localization. Nevertheless, inactive CPEB accumulates in granules in terminal dendritic branches, supporting the hypothesis that upon activation CPEB and its mRNA cargo are released from granules and are then available for dendritic translation.

Figure 2: Time-lapse of CPEB–CFP/YFP expressing tectal neuron. One minute interval time-lapse frames made of the dendrites of the CPEB–CFP/YFP-expressing cell in Figure 1A. The time-lapse series focuses on the boxed area of the dendrites. Arrows indicate the progressive history of locations of one punctum over the 12 min, with the current position in yellow, past positions in white. Smaller insert illustrates the rapid fusing and splitting of puncta. Scale bar = 10 μm. See also Supplementary Video 1.

Mentions:
We acquired 1-min interval time-lapse series of dendrites to see if RNP granules are mobile. Similar to reports of RNP granule mobility in cultured neurons (Knowles and Kosik, 1997; Huang et al., 2003; Antar et al., 2004, 2005; Dynes and Steward, 2007), our results revealed that the CPEB and delCPEB puncta largely exhibited oscillatory movements, with no net change in position over time (Figures 2–4 and Supplementary Videos 1–3). The high density of puncta in the branches combined with their oscillatory movements often resulted in image sequences where two or more puncta appear to fuse together and split apart, precluding their unambiguous identification through the time series even with the 1-min interval time points (Figure 2, inserts and Figure 3). Despite this, obvious directional movements of some puncta could be resolved across time points over the series (arrows, Figure 2). The mobility of the CPEB- and delCPEB-containing RNP granules was not detectably different from one another. In the relatively shorter terminal branches puncta did not display clear directional movements (Figures 2–4), but new puncta appear and coalesce and change intensity over time (Figures 3–4 and Supplementary Videos 2 and 3). These data suggest that the majority of RNP granules imaged in neurons in the intact animal undergo relatively small local movements and that a minority exhibit directional movements.

Figure 2: Time-lapse of CPEB–CFP/YFP expressing tectal neuron. One minute interval time-lapse frames made of the dendrites of the CPEB–CFP/YFP-expressing cell in Figure 1A. The time-lapse series focuses on the boxed area of the dendrites. Arrows indicate the progressive history of locations of one punctum over the 12 min, with the current position in yellow, past positions in white. Smaller insert illustrates the rapid fusing and splitting of puncta. Scale bar = 10 μm. See also Supplementary Video 1.

Mentions:
We acquired 1-min interval time-lapse series of dendrites to see if RNP granules are mobile. Similar to reports of RNP granule mobility in cultured neurons (Knowles and Kosik, 1997; Huang et al., 2003; Antar et al., 2004, 2005; Dynes and Steward, 2007), our results revealed that the CPEB and delCPEB puncta largely exhibited oscillatory movements, with no net change in position over time (Figures 2–4 and Supplementary Videos 1–3). The high density of puncta in the branches combined with their oscillatory movements often resulted in image sequences where two or more puncta appear to fuse together and split apart, precluding their unambiguous identification through the time series even with the 1-min interval time points (Figure 2, inserts and Figure 3). Despite this, obvious directional movements of some puncta could be resolved across time points over the series (arrows, Figure 2). The mobility of the CPEB- and delCPEB-containing RNP granules was not detectably different from one another. In the relatively shorter terminal branches puncta did not display clear directional movements (Figures 2–4), but new puncta appear and coalesce and change intensity over time (Figures 3–4 and Supplementary Videos 2 and 3). These data suggest that the majority of RNP granules imaged in neurons in the intact animal undergo relatively small local movements and that a minority exhibit directional movements.

Bottom Line:
In dendrites, the distributions of the active and inactive CPEB-containing RNP granules do not differ; the RNP granules are dense and their positions do not correlate with sites of rapid dendritic branch dynamics or the eventual fate of the dendritic branches.Because CPEB's sensitivity to activity-dependent signaling does not alter its dendritic distribution, it indicates that active sites in the dendritic arbor are not targeted for RNP granule localization.Nevertheless, inactive CPEB accumulates in granules in terminal dendritic branches, supporting the hypothesis that upon activation CPEB and its mRNA cargo are released from granules and are then available for dendritic translation.

ABSTRACTCytoplasmic Polyadenylation Element Binding protein (CPEB) is an RNA binding protein involved in dendritic delivery of mRNA and activity-dependent, polyadenylation-induced translation of mRNAs in the dendritic arbor. CPEB affects learning and memory and impacts neuronal morphological and synaptic plasticity. In neurons, CPEB is concentrated in ribonucleoprotein (RNP) granules that distribute throughout the dendritic arbor and localize near synapses, suggesting that the trafficking of RNP granules is important for CPEB function. We tagged full-length CPEB and an inactive mutant CPEB with fluorescent proteins, then imaged rapid dendritic branch dynamics and RNP distribution using two-photon time-lapse microscopy of neurons in the optic tectum of living Xenopus laevis tadpoles. Though the inactive CPEB mutant transports mRNA in the dendritic arbor, its expression interferes with CPEB-dependent translation because it is incapable of activity-triggered mRNA polyadenylation. In dendrites, the distributions of the active and inactive CPEB-containing RNP granules do not differ; the RNP granules are dense and their positions do not correlate with sites of rapid dendritic branch dynamics or the eventual fate of the dendritic branches. Because CPEB's sensitivity to activity-dependent signaling does not alter its dendritic distribution, it indicates that active sites in the dendritic arbor are not targeted for RNP granule localization. Nevertheless, inactive CPEB accumulates in granules in terminal dendritic branches, supporting the hypothesis that upon activation CPEB and its mRNA cargo are released from granules and are then available for dendritic translation.